Isabella Graef

All Publications

• AG10 inhibits amyloidogenesis and cellular toxicity of the familial amyloid cardiomyopathy-associated V122I transthyretin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Penchala, S. C., Connelly, S., Wang, Y., Park, M. S., Zhao, L., Baranczak, A., Rappley, I., Vogel, H., Liedtke, M., Witteles, R. M., Powers, E. T., Reixach, N., Chan, W. K., Wilson, I. A., Kelly, J. W., Graef, I. A., Alhamadsheh, M. M. 2013; 110 (24): 9992-9997

  Abstract

  The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy.

  View details for DOI 10.1073/pnas.1300761110

  View details for Web of Science ID 000320930100085

  View details for PubMedID 23716704

• Design of beta-Amyloid Aggregation Inhibitors from a Predicted Structural Motif JOURNAL OF MEDICINAL CHEMISTRY Novick, P. A., Lopes, D. H., Branson, K. M., Esteras-Chopo, A., Graef, I. A., Bitan, G., Pande, V. S. 2012; 55 (7): 3002-3010

  Abstract

  Drug design studies targeting one of the primary toxic agents in Alzheimer's disease, soluble oligomers of amyloid β-protein (Aβ), have been complicated by the rapid, heterogeneous aggregation of Aβ and the resulting difficulty to structurally characterize the peptide. To address this, we have developed [Nle(35), D-Pro(37)]Aβ(42), a substituted peptide inspired from molecular dynamics simulations which forms structures stable enough to be analyzed by NMR. We report herein that [Nle(35), D-Pro(37)]Aβ(42) stabilizes the trimer and prevents mature fibril and β-sheet formation. Further, [Nle(35), D-Pro(37)]Aβ(42) interacts with WT Aβ(42) and reduces aggregation levels and fibril formation in mixtures. Using ligand-based drug design based on [Nle(35), D-Pro(37)]Aβ(42), a lead compound was identified with effects on inhibition similar to the peptide. The ability of [Nle(35), D-Pro(37)]Aβ(42) and the compound to inhibit the aggregation of Aβ(42) provides a novel tool to study the structure of Aβ oligomers. More broadly, our data demonstrate how molecular dynamics simulation can guide experiment for further research into AD.

  View details for DOI 10.1021/jm201332p

  View details for Web of Science ID 000302591100010

  View details for PubMedID 22420626

• Potent Kinetic Stabilizers That Prevent Transthyretin-Mediated Cardiomyocyte Proteotoxicity SCIENCE TRANSLATIONAL MEDICINE Alhamadsheh, M. M., Connelly, S., Cho, A., Reixach, N., Powers, E. T., Pan, D. W., Wilson, I. A., Kelly, J. W., Graef, I. A. 2011; 3 (97)

  Abstract

  A valine-to-isoleucine mutation at position 122 of the serum protein transthyretin (TTR), found in 3 to 4% of African Americans, alters its stability, leading to amyloidogenesis and cardiomyopathy. In addition, 10 to 15% of individuals older than 65 years develop senile systemic amyloidosis and cardiac TTR deposits because of wild-type TTR amyloidogenesis. Although several drugs are in development, no approved therapies for TTR amyloid cardiomyopathy are yet available, so the identification of additional compounds that prevent amyloid-mediated cardiotoxicity is needed. To this aim, we developed a fluorescence polarization-based high-throughput screen and used it to identify several new chemical scaffolds that target TTR. These compounds were potent kinetic stabilizers of TTR and prevented TTR tetramer dissociation, partial unfolding, and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR. High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. These compounds effectively inhibited the proteotoxicity of V122I-TTR toward human cardiomyocytes. Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is a well-studied inhibitor of TTR aggregation, and may be promising leads for the treatment or prevention of TTR-mediated cardiomyopathy.

  View details for DOI 10.1126/scitranslmed.3002473

  View details for Web of Science ID 000294207000005

  View details for PubMedID 21865539

• Calcineurin/NFAT Signaling Is Required for Neuregulin-Regulated Schwann Cell Differentiation SCIENCE Kao, S., Wu, H., Xie, J., Chang, C., Ranish, J. A., Graef, I. A., Crabtree, G. R. 2009; 323 (5914): 651-654

  Abstract

  Schwann cells develop from multipotent neural crest cells and form myelin sheaths around axons that allow rapid transmission of action potentials. Neuregulin signaling through the ErbB receptor regulates Schwann cell development; however, the downstream pathways are not fully defined. We find that mice lacking calcineurin B1 in the neural crest have defects in Schwann cell differentiation and myelination. Neuregulin addition to Schwann cell precursors initiates an increase in cytoplasmic Ca2+, which activates calcineurin and the downstream transcription factors NFATc3 and c4. Purification of NFAT protein complexes shows that Sox10 is an NFAT nuclear partner and synergizes with NFATc4 to activate Krox20, which regulates genes necessary for myelination. Our studies demonstrate that calcineurin and NFAT are essential for neuregulin and ErbB signaling, neural crest diversification, and differentiation of Schwann cells.

  View details for DOI 10.1126/science.1166562

  View details for Web of Science ID 000262862800048

  View details for PubMedID 19179536

• Bursting into the Nucleus SCIENCE SIGNALING Crabtree, G. R., Graef, I. A. 2008; 1 (51)

  Abstract

  An increase in extracellular Ca(2+) induces the nuclear localization of the Crz1 transcription factor and the activation of target genes in yeast. A recent study indicates that nuclear entry occurs in short stochastic bursts that are unsynchronized within the population of cells. The frequency but not the amplitude of the bursts is controlled by Ca(2+). Modulation of the frequency of the burst coordinates aspects of expression of Crz target genes.

  View details for DOI 10.1126/scisignal.151pe54

  View details for Web of Science ID 000207497900002

  View details for PubMedID 19109237

• Regulation of dendritic development by NeuronSpecific chromatin remodeling complexes NEURON Wu, J. I., Lessard, J., Olave, I. A., Qiu, Z., Ghosh, A., Graef, I. A., Crabtree, G. R. 2007; 56 (1): 94-108

  Abstract

  The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53b's role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.

  View details for DOI 10.1016/j.neuron.2007.08.021

  View details for Web of Science ID 000250289800013

  View details for PubMedID 17920018

• An essential switch in subunit composition of a chromatin remodeling complex during neural development NEURON Lessard, J., Wu, J. I., Ranish, J. A., Wan, M., Winslow, M. M., Staahl, B. T., Wu, H., Aebersold, R., Graef, I. A., Crabtree, G. R. 2007; 55 (2): 201-215

  Abstract

  Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.

  View details for DOI 10.1016/j.neuron.2007.06.019

  View details for Web of Science ID 000248635800006

  View details for PubMedID 17640523

  View details for PubMedCentralID PMC2674110

• NFAT signaling and the invention of vertebrates TRENDS IN CELL BIOLOGY Wu, H., Peisley, A., Graef, I. A., Crabtree, G. R. 2007; 17 (6): 251-260

  Abstract

  The calcium/calcineurin-dependent NFATc family is thought to have arisen following the recombination of an ancient precursor with a Rel domain about 500 million years ago, producing a new group of signaling and transcription factors (the NFATc genes) found only in the genomes of vertebrates. Cell biological, genetic and biochemical evidence indicates that the circuitry of this pathway is well suited for intercalation with older pathways. We propose that this recombination enabled Ca(2+) signals to be redirected to a new transcriptional program, which provided part of the groundwork for vertebrate morphogenesis and organogenesis. This notion predicts that calcineurin-NFAT signaling would be essential for much of vertebrate development. We review recent evidence supporting this prediction and propose a systematic approach to explore aspects of vertebrate morphogenesis.

  View details for DOI 10.1016/j.tcb.2007.04.006

  View details for Web of Science ID 000247508200001

  View details for PubMedID 17493814

• Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhou, X., Babu, J. R., da Silva, S., Shu, Q., Graef, I. A., Oliver, T., Tomoda, T., Tani, T., Wooten, M. W., Wang, F. 2007; 104 (14): 5842-5847

  Abstract

  The molecular mechanism and significance of endocytic processes involved in directional axon elongation are not well understood. The Unc-51 family of serine/threonine kinases was shown to be important for axon growth and was also linked to endocytosis, providing an entry point to study this problem. We found that mouse Unc-51-like kinase 1/2 (Ulk1/2) proteins are localized to vesicular structures in growth cones of mouse spinal sensory neurons. RNAi-mediated knockdown of Ulk1 and/or Ulk2 resulted in impaired endocytosis of nerve growth factor (NGF), excessive axon arborization, and severely stunted axon elongation. The evidence also indicates that Ulk1/2 mediates a non-clathrin-coated endocytosis in sensory growth cones. Interestingly, NGF can induce the interaction of Ulk1 with TrkA receptor complexes through promoting K63-polyubiquitination of Ulk1 and binding of Ulk1 to the scaffolding protein p62. These results and additional studies suggest that Ulk1/2 proteins regulate filopodia extension and neurite branching during sensory axon outgrowth, probably through regulating TrkA receptor trafficking and signaling.

  View details for DOI 10.1073/pnas.0701402104

  View details for Web of Science ID 000245657600027

  View details for PubMedID 17389358

  View details for PubMedCentralID PMC1851579

• Role of transcription factor NFAT in glucose and insulin homeostasis MOLECULAR AND CELLULAR BIOLOGY Yang, T. T., Suk, H. Y., Yang, X., Olabisi, O., Yu, R. Y., Durand, J., Jelicks, L. A., Kim, J., Scherer, P. E., Wang, Y., Feng, Y., Rossetti, L., Graef, I. A., Crabtree, G. R., Chow, C. 2006; 26 (20): 7372-7387

  Abstract

  Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2-/- Nfatc4-/- compound disruption exhibit defects in fat accumulation and are lean. Nfatc2-/- Nfatc4-/- mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2-/- Nfatc4-/- mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.

  View details for DOI 10.1128/MCB.00580-06

  View details for Web of Science ID 000241252300003

  View details for PubMedID 16908540

  View details for PubMedCentralID PMC1636854

• NFAT dysregulation by increased dosage of DSCR1 and DYRK1A on chromosome 21 Nature Arron*, W. P., Chang, Wu, Gao, Neilson, Chen, Heit, Kim, Yamasaki, Miyakawa, Francke, Graef*#, Crabtree# 2006; 441 (June 1): 595-600
• 'Nature-inspired' drug-protein complexes as inhibitors of A beta aggregation Conference on Molecular Mechanisms of Neurodegeneration Bose, M., Gestwicki, J. E., Devasthali, V., Crabtree, G. R., Graef, I. A. PORTLAND PRESS LTD. 2005: 543–547

  Abstract

  Protein-protein interactions are a regulatory mechanism for a number of physiological and pathological cellular processes. Neurodegenerative diseases, such as AD (Alzheimer's disease), are associated with the accelerated production or delayed clearance of protein aggregates. Hence, inhibition of pathologic protein-protein interactions is a very attractive mechanism for drug development. This review focuses on a novel therapeutic strategy to inhibit the de novo formation of protein aggregates. Inspired by strategies used in Nature and optimized over millions of years of evolution, we have created a bifunctional molecule [SLF (synthetic ligand for FK506-binding protein)-CR (Congo Red)] that is able to block Abeta (amyloid beta) aggregation by borrowing the surface and steric bulk of a cellular chaperone.

  View details for Web of Science ID 000231345700001

  View details for PubMedID 16042540

• Recruitment of the extracellular signal-regulated kinase/ribosomal S6 kinase signaling pathway to the NFATc4 transcription activation complex MOLECULAR AND CELLULAR BIOLOGY Yang, T. T., Xiong, Q. F., Graef, I. A., Crabtree, G. R., Chow, C. W. 2005; 25 (3): 907-920

  Abstract

  Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calcium-mediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser(676) and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser(676) is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.

  View details for DOI 10.1128/MCB.25.3.907-920.2005

  View details for Web of Science ID 000226652900005

  View details for PubMedID 15657420

• Harnessing chaperones to generate small-molecule inhibitors of amyloid beta aggregation SCIENCE Gestwicki, J. E., Crabtree, G. R., Graef, I. A. 2004; 306 (5697): 865-869

  Abstract

  Protein aggregation is involved in the pathogenesis of neurodegenerative diseases and hence is considered an attractive target for therapeutic intervention. However, protein-protein interactions are exceedingly difficult to inhibit. Small molecules lack sufficient steric bulk to prevent interactions between large peptide surfaces. To yield potent inhibitors of beta-amyloid (Abeta) aggregation, we synthesized small molecules that increase their steric bulk by binding to chaperones but also have a moiety available for interaction with Abeta. This strategy yields potent inhibitors of Abeta aggregation and could lead to therapeutics for Alzheimer's disease and other forms of neurodegeneration.

  View details for Web of Science ID 000224969400045

  View details for PubMedID 15514157

• A field of myocardial-endocardial NFAT signaling underlies heart valve morphogenesis CELL CHANG, C. P., Neilson, J. R., Bayle, J. H., Gestwicki, J. E., Kuo, A., Stankunas, K., Graef, I. A., Crabtree, G. R. 2004; 118 (5): 649-663

  Abstract

  The delicate leaflets that make up vertebrate heart valves are essential for our moment-to-moment existence. Abnormalities of valve formation are the most common serious human congenital defect. Despite their importance, relatively little is known about valve development. We show that the initiation of heart valve morphogenesis in mice requires calcineurin/NFAT to repress VEGF expression in the myocardium underlying the site of prospective valve formation. This repression of VEGF at E9 is essential for endocardial cells to transform into mesenchymal cells. Later, at E11, a second wave of calcineurin/NFAT signaling is required in the endocardium, adjacent to the earlier myocardial site of NFAT action, to direct valvular elongation and refinement. Thus, NFAT signaling functions sequentially from myocardium to endocardium within a valvular morphogenetic field to initiate and perpetuate embryonic valve formation. This mechanism also operates in zebrafish, indicating a conserved role for calcineurin/NFAT signaling in vertebrate heart valve morphogenesis.

  View details for Web of Science ID 000223730300014

  View details for PubMedID 15339668

• Neurotrophins and netrins require calcineurin/NFAT signaling to stimulate outgrowth of embryonic axons CELL Graef, I. A., Wang, F., Charron, F., Chen, L., Neilson, J., Tessier-Lavigne, M., Crabtree, G. R. 2003; 113 (5): 657-670

  Abstract

  Axon outgrowth is the first step in the formation of neuronal connections, but the pathways that regulate axon extension are still poorly understood. We find that mice deficient in calcineurin-NFAT signaling have dramatic defects in axonal outgrowth, yet have little or no defect in neuronal differentiation or survival. In vitro, sensory and commissural neurons lacking calcineurin function or NFATc2, c3, and c4 are unable to respond to neurotrophins or netrin-1 with efficient axonal outgrowth. Neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons. These data indicate that the ability of these embryonic axons to respond to growth factors with rapid outgrowth requires activation of calcineurin/NFAT signaling by these factors. The precise parsing of signals for elongation turning and survival could allow independent control of these processes during development.

  View details for Web of Science ID 000183397600015

  View details for PubMedID 12787506

• NFAT signaling in vertebrate development CURRENT OPINION IN GENETICS & DEVELOPMENT Graef, I. A., Chen, F., Crabtree, G. R. 2001; 11 (5): 505-512

  Abstract

  NFATc proteins transduce Ca(2+) signals to the nucleus and then pair with other proteins on DNA to generate NFAT complexes that activate transcription in response to both electrical and tyrosine kinase signaling. The four NFATc genes arose at the origin of vertebrates, implying that they have evolved for the development of vertebrate-specific functions, such as a complex nervous system, a recombinational immune system, and a vascular system with a complex heart. These speculations are borne out by studies of mice with null mutations in the different family members.

  View details for Web of Science ID 000171477500003

  View details for PubMedID 11532391

• Signals transduced by Ca2+/calcineurin and NFATc3/c4 pattern the developing vasculature CELL Graef, I. A., Chen, F., Chen, L., Kuo, A., Crabtree, G. R. 2001; 105 (7): 863-875

  Abstract

  Vascular development requires an orderly exchange of signals between growing vessels and their supporting tissues, but little is known of the intracellular signaling pathways underlying this communication. We find that mice with disruptions of both NFATc4 and the related NFATc3 genes die around E11 with generalized defects in vessel assembly as well as excessive and disorganized growth of vessels into the neural tube and somites. Since calcineurin is thought to control nuclear localization of NFATc proteins, we introduced a mutation into the calcineurin B gene that prevents phosphatase activation by Ca(2+) signals. These CnB mutant mice exhibit vascular developmental abnormalities similar to the NFATc3/c4 null mice. We show that calcineurin function is transiently required between E7.5 and E8.5. Hence, early calcineurin/NFAT signaling initiates the later cross-talk between vessels and surrounding tissues that pattern the vasculature.

  View details for Web of Science ID 000169664300007

  View details for PubMedID 11439183

• Evolutionary relationships among Rel domains indicate functional diversification by recombination PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Graef, I. A., Gastier, J. M., FRANCKE, U., Crabtree, G. R. 2001; 98 (10): 5740-5745

  Abstract

  The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function of complex multidomain proteins in mammals produced by accretion of domains. With this objective, we have determined the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (i) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (ii) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (iii) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca(2+)/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. On the other hand, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system.

  View details for Web of Science ID 000168623300067

  View details for PubMedID 11344309

• Jun N-terminal kinase 2 modulates thymocyte apoptosis and T cell activation through c-Jun and nuclear factor of activated T cell (NF-AT) PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Behrens, A., Sabapathy, K., Graef, I., Cleary, M., Crabtree, G. R., Wagner, E. F. 2001; 98 (4): 1769-1774

  Abstract

  The Jun N-terminal kinases (JNKs) recently have been shown to be required for thymocyte apoptosis and T cell differentiation and/or proliferation. To investigate the molecular targets of JNK signaling in lymphoid cells, we used mice in which the serines phosphorylated by JNK in c-Jun were replaced by homologous recombination with alanines (junAA mice). Lymphocytes from these mice showed no phosphorylation of c-Jun in response to activation stimuli, whereas c-Jun was rapidly phosphorylated in wild-type cells. Despite the fact that c-jun is essential for early development, junAA mice develop normally; however, c-Jun N-terminal phosphorylation was required for efficient T cell receptor-induced and tumor necrosis factor-alpha-induced thymocyte apoptosis. In contrast, c-Jun phosphorylation by JNK is not required for T cell proliferation or differentiation. Because jnk2-/- T cells display a proliferation defect, we concluded that JNK2 must have other substrates required for lymphocyte function. Surprisingly, jnk2-/- T cells showed reduced NF-AT DNA-binding activity after activation. Furthermore, overexpression of JNK2 in Jurkat T cells strongly enhanced NF-AT-dependent transcription. These results demonstrate that JNK signaling differentially uses c-Jun and NF-AT as molecular effectors during thymocyte apoptosis and T cell proliferation.

  View details for Web of Science ID 000166949200083

  View details for PubMedID 11172026

• c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation JOURNAL OF EXPERIMENTAL MEDICINE Sabapathy, K., Kallunki, T., David, J. P., Graef, I., Karin, M., Wagner, E. F. 2001; 193 (3): 317-328

  Abstract

  Apoptotic and mitogenic stimuli activate c-Jun NH2-terminal kinases (JNKs) in T cells. Although T cells express both JNK1 and JNK2 isozymes, the absence of JNK2 alone can result in resistance to anti-CD3-induced thymocyte apoptosis and defective mature T cell proliferation. Similar defects in thymocyte apoptosis and mature T cell proliferation, the latter due to reduced interleukin 2 production, are also caused by JNK1 deficiency. Importantly, T cell function was compromised in Jnk1(+/-)Jnk2(+/-) double heterozygous mice, indicating that JNK1 and JNK2 play similar roles in regulating T cell function. The reduced JNK dose results in defective c-Jun NH2-terminal phosphorylation in thymocytes but not in peripheral T cells, in which nuclear factors of activated T cells (NK-ATs)-DNA binding activity is affected. Thus, JNK1 and JNK2 control similar functions during T cell maturation through differential targeting of distinct substrates.

  View details for Web of Science ID 000166902900005

  View details for PubMedID 11157052

• L-type calcium channels and GSK-3 regulate the activity of NF-ATc4 in hippocampal neurons NATURE Graef, I. A., Mermelstein, P. G., Stankunas, K., Neilson, J. R., Deisseroth, K., Tsien, R. W., Crabtree, G. R. 1999; 401 (6754): 703-708

  Abstract

  The molecular basis of learning and memory has been the object of several recent advances, which have focused attention on calcium-regulated pathways controlling transcription. One of the molecules implicated by pharmacological, biochemical and genetic approaches is the calcium/calmodulin-regulated phosphatase, calcineurin. In lymphocytes, calcineurin responds to specific calcium signals and regulates expression of several immediate early genes by controlling the nuclear import of the NF-ATc family of transcription factors. Here we show that NF-ATc4/NF-AT3 in hippocampal neurons can rapidly translocate from cytoplasm to nucleus and activate NF-AT-dependent transcription in response to electrical activity or potassium depolarization. The calcineurin-mediated translocation is critically dependent on calcium entry through L-type voltage-gated calcium channels. GSK-3 can phosphorylate NF-ATc4, promoting its export from the nucleus and antagonizing NF-ATc4-dependent transcription. Furthermore, we show that induction of the inositol 1,4,5-trisphosphate receptor type 1 is controlled by the calcium/calcineurin/NF-ATc pathway. This provides a new perspective on the function of calcineurin in the central nervous system and indicates that NF-AT-mediated gene expression may be involved in the induction of hippocampal synaptic plasticity and memory formation.

  View details for Web of Science ID 000083207400058

  View details for PubMedID 10537109

• Signaling through calcium, calcineurin, and NF-AT in lymphocyte activation and development 64th Symposia: Signaling and Gene Expression in the Immune System Stankunas, K., Graef, I. A., Neilson, J. R., Park, S. H., Crabtree, G. R. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1999: 505–516

  View details for Web of Science ID 000087225400061

  View details for PubMedID 11232327

• Defects in actin-cap formation in Vav-deficient mice implicate an actin requirement for lymphocyte signal transduction CURRENT BIOLOGY Holsinger, L. J., Graef, I. A., Swat, W., Chi, T., Bautista, D. M., Davidson, L., Lewis, R. S., Alt, F. W., Crabtree, G. R. 1998; 8 (10): 563-572

  Abstract

  Antigen-receptor interactions on lymphocytes result in local clustering of actin, receptors and signaling molecules into an asymmetric membrane structure termed a cap. Although actin polymerization is known to be required, the mechanisms underlying cap formation are unclear. We have studied the events underlying cap formation using mice bearing a null mutation in vav (vav-/-), a gene that encodes a guanine-nucleotide exchange factor for the GTPase Rac.Lymphocytes from vav-/- mice failed to form T-cell receptor caps following activation and had a defective actin cytoskeleton. The vav-/- T cells were deficient in interleukin-2 (IL-2) production and proliferation, and the peak of Ca2+ mobilization was reduced although of normal duration. Activation of Jun N-terminal kinase or stress-activated kinase (JNK or SAPK) and mitogen-activated protein kinase (MAPK) and the induction of the transcription factor NF-ATc1 and egr-1 genes was normal. Despite the reduced Ca2+ mobilization, translocation of cytoplasmic NF-ATc to the nucleus was normal, reflecting that the lower levels of Ca2+ in vav-/- cells were still sufficient to activate calcineurin. Treatment of lymphocytes with cytochalasin D, which blocks actin polymerization, inhibited cap formation and produced defects in signaling and IL-2 transcriptional induction in response to antigen-receptor signaling that were nearly identical to those seen in vav-/- cells. In transfection studies, either constitutively active Vav or Rac could complement constitutively active calcineurin to activate NF-AT-dependent transcription.These results indicate that Vav is required for cap formation in lymphocytes. Furthermore, the correlation between cap formation, IL-2 production and proliferation supports the hypothesis that an actin-dependent pathway is a source of specialized growth regulatory signals.

  View details for Web of Science ID 000073590300014

  View details for PubMedID 9601640

• Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70 EMBO JOURNAL Graef, I. A., Holsinger, L. J., Diver, S., Schreiber, S. L., Crabtree, G. R. 1997; 16 (18): 5618-5628

  Abstract

  Signaling by the antigen receptor of T lymphocytes initiates different developmental transitions, each of which require the tyrosine kinase ZAP70. Previous studies with agonist and antagonist peptides have indicated that ZAP70 might respond differently to different structures of the TCR-CD3 complex induced by bound peptides. The roles of membrane proximity and orientation in activation of ZAP70 signaling were explored using synthetic ligands and their binding proteins designed to produce different architectures of membrane-bound complexes composed of ZAP70 fusion proteins. Transient membrane recruitment of physiological levels of ZAP70 with the membrane-permeable synthetic ligand FK1012A leads to rapid phosphorylation of ZAP70 and activation of the ras/MAPK and Ca2+/calcineurin signaling pathways. ZAP70 SH2 domains are not required for signaling when the kinase is artifically recruited to the membrane, indicating that the SH2 domains function solely in recruitment and not in kinase activation. Using additional synthetic ligands and their binding proteins that recruit ZAP70 equally well but orient it at the cell membrane in different ways, we define a requirement for a specific presentation of ZAP70 to its downstream targets. These results provide a mechanism by which ZAP70, bound to the phosphorylated receptor, could discriminate between conformational changes induced by the binding of different MHC-peptide complexes to the antigen receptor and introduce an approach to exploring the role of spatial orientation of signaling complexes in living cells.

  View details for Web of Science ID A1997XY11200015

  View details for PubMedID 9312021

  View details for PubMedCentralID PMC1170194

• The transcriptional paradox: Octamer factors and B and T cells SCIENCE Graef, I. A., Crabtree, G. R. 1997; 277 (5323): 193-194

  View details for Web of Science ID A1997XK41800030

  View details for PubMedID 9235633

• The mechanism of action of cyclosporin A and FK506 Symposium on New Frontiers in the Immunoregulation of Allergic and Autoimmune Diseases Ho, S., Clipstone, N., Timmermann, L., Northrop, J., Graef, I., Fiorentino, D., Nourse, J., Crabtree, G. R. ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS. 1996: S40–S45

  Abstract

  The immunosuppressants cyclosporin A (CsA), FK506, and rapamycin suppress the immune response by inhibiting evolutionary conserved signal transduction pathways. CsA, FK506, and rapamycin bind to their intracellular receptors, immunophilins, creating composite surfaces that block the activity of specific targets. For CsA/cyclophilin and FK506/FKBP the target is calcineurin. Because of the large surface area of interaction of the drug-immunophilin complex with calcineurin, FK506 and CsA have a specificity for their biologic targets that is equivalent to growth factor-receptor interactions. To date, all the therapeutic as well as toxic effects of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of the action of calcineurin results in a complete block in the translocation of the cytosolic component of the nuclear factor of activated T cells (NF-AT), resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin (IL-4) and CD40 ligand as well as those necessary for T-cell proliferation such as IL-2. The purpose of this article is to illustrate the means by which these drugs produce immunosuppression.

  View details for Web of Science ID A1996VG75000006

  View details for PubMedID 8811062

• A GENERAL STRATEGY FOR PRODUCING CONDITIONAL ALLELES OF SRC-LIKE TYROSINE KINASES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Spencer, D. M., Graef, I., AUSTIN, D. J., Schreiber, S. L., Crabtree, G. R. 1995; 92 (21): 9805-9809

  Abstract

  The Src-like tyrosine kinases require membrane localization for transformation and probably for their normal role in signal transduction. We utilized this characteristic to prepare Src-like tyrosine kinases that can be readily activated with the rationally designed chemical inducer of dimerization FK1012. Dimerization of cytoplasmic Src-like tyrosine kinases was not sufficient for signaling, but their recruitment to the plasma membrane led to the rapid activation of transcription factors identical to those regulated by crosslinking the antigen receptor. Moreover, recruitment of activated Src-like kinases to the membrane replaced signaling by the T-lymphocyte antigen receptor complex, leading to the activation of both the Ras/protein kinase C and Ca2+/calcineurin pathways normally activated by antigen receptor signaling. Since these chemical inducers of dimerization are cell permeable, this approach permits the production of conditional alleles of any of the Src-like tyrosine kinases, thereby allowing a delineation of their developmental roles.

  View details for Web of Science ID A1995RZ07200077

  View details for PubMedID 7568222